Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Reexamination Certificate
1998-12-02
2001-01-09
Brusca, John S. (Department: 1631)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C528S327000, C528S272000, C528S289000, C530S333000
Reexamination Certificate
active
06171819
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to the catalysis of chemical reactions, and more particularly to the catalysis of acyl transfer reactions.
BACKGROUND OF THE INVENTION
So-called acyl transfer reactions involve the transfer of an acyl group (the residue of an organic acid after removal of the carboxyl hydroxy group) either internally within a chemical species or from one chemical species to another. Examples are amide formation, transesterification and hydrolysis.
It is well known that acyl transfer reactions may be catalyzed by imidazole in aqueous solution, the imidazole, which is a strong nucleophile, forming an intermediary reactive complex with the acyl group. Also polymer-supported imidazoles have been used as acyl transfer catalysts (see e.g. Skjujins, A., et al., Latv. PSR Zinat. Akad. Vestis, Kim. Ser. 1988 (6), 720-5).
It has further been shown that small peptides containing a histidine (His) residue (an amino acid which contains an imidazolyl group) may have hydrolytic activity.
Recent progress in designing structural proteins and peptides have resulted in the preparation of several peptides with substantial catalytic activity (W. F. DeGrado, Nature, 365, 488 (1993). For example, K. Johnsson et al., Nature, 365, 530 (1993) disclose a short self-associating Leu-Lys-rich helical peptide that accelerates the rate of decarboxylation by means of a Schiff's base intermediate between a substrate of oxaloacetate and an amine with an electrostatically depressed acid constant (K
a
). It is mentioned that the secondary structure is important for the activity.
The present invention provides improvements in designed catalytic structures including an imidazole-based catalytic function.
SUMMARY OF THE INVENTION
According to the present invention it has been found that the above described imidazole induced catalytic activity in acyl transfer reactions may be increased considerably if the imidazolyl moiety is provided in a chemical structure flanked on one or both sides by a group of such a nature and position that it is capable of stabilizing the transition complex formed between the imidazolyl group and the acyl group in question. To accomplish such a stabilization, the flanking group or groups should be capable of molecular interaction with the acyl complex, such as by hydrogen bonding, electrostatic or hydrophobic interactions or van der Waal forces (intramolecular polarization).
The increased catalytic activity may be used in combination with intermolecular as well as intramolecular reactions in solution, with and without stereospecifity. In the latter case it is possible to make site selective functionalization of peptides and other molecules. Such site selective functionalization will inter alia permit site selective immobilization of molecules, such as biomolecules, e.g. antibodies or other proteins or polypeptides.
One of the objects of the invention is to provide an improved method of performing an acyl transfer type reaction using an imidazole based catalyst.
In a first aspect of the invention, there is therefore provided an improved method of performing a chemical reaction involving an acyl transfer mechanism in the presence of an imidazole-based catalyst which can form a transition complex with the acyl group. The method is characterized in that the imidazole function is provided by a chemical structure element comprising an imidazolyl group flanked on one or both sides by a group capable of stabilizing the transition complex by molecular interaction with the acyl group. This molecular interaction may be selected from hydrogen bonding, electrostatic interaction and hydrophobic interaction.
In a preferred embodiment of the method, the chemical structure element constitutes or is part of a larger structure having a functional group in such a neighboring position that it can be site-specifically functionalized through the acyl transfer via the above intermediary complex.
Another object of the invention is to provide a chemical structure element with improved capability of catalyzing an acyl transfer reaction.
In a second aspect of the invention, there is therefore provided a chemical structure element comprising backbone structure with a pendant imidazole function, which element is characterized in that the imidazole function is flanked on one or both sides on said backbone structure by a pendant group capable of stabilizing the transition complex by molecular interaction with the acyl group.
In one embodiment, the structure element is a molecule, such as a peptide or protein, comprising a function in such a neighboring position that it can be site-specifically functionalized through the acyl transfer via the above intermediary complex.
Yet another object of the invention is to provide a method of producing by genetic engineering a protein or peptide constituting or comprising a structure element having an imidazole function flanked on one or both sides by a transition complex stabilizing group.
In a third aspect, the invention therefore provides a method of producing a protein or peptide which constitutes or comprises an imidazole function-containing structure element as defined above, which method comprises transforming a host organism with a recombinant DNA construct comprising a vector and a DNA sequence encoding said protein or peptide, culturing the host organism to express said protein or peptide, and isolating the latter from the culture.
In a preferred embodiment of the method, the structure element comprises a functional group in a such a neighboring position to the imidazole function that the function can be site-specifically functionalized through acyl transfer catalyzed by the imidazole function.
Still another object of the invention is to provide a vector comprising a nucleic acid sequence encoding the above protein or peptide.
In a fourth aspect, the invention therefore provides a recombinant DNA construct comprising a vector and a DNA sequence encoding a protein or peptide which constitutes or comprises an imidazole function-containing structure element as defined above.
In a preferred embodiment of the vector, the DNA sequence also encodes a specific functional group in a such a neighboring position to the imidazole function that the functional group can be site-specifically functionalized through acyl transfer catalyzed by the imidazole function.
REFERENCES:
Guthrie, JP, et al, “Hydration of acylimidazoles: tetrahedral intermediates in acylimidazole hydrolysis and nucleophilic attack by imidazole on esters. The question of concerted mechanisms for acyl transfers”, Can J Chem vol 65, 1987, pp 1951-1969.
Visser, HGJ, et al, “Synthesis of polymers of isocyanides derived from tripeptides containing imidazolyl, carboxyl, and hydroxymethyl groups”, J Org Chem, 1985, 50, 3133-3137.
Hajdu, J, et al, “Catalytic mechanisms of acyl transfer reactions in dipolar aprotic media 2. electrophilic activation of the carbonyl group by quarternary alkylammonium and imidazolium functions”, J Am Chem Soc, 1981, 103, 6192-6197.
Goren, HJ, et al, “Poly(L-histidyl-L-alanyl-aphal-L-Glutamic acid). II. Catalysis of p-Nitrophenyl acetate hydrolysis”, Biopolymers, vol. 17, 1978, 1679-1692.
Lee, S.G., et al, “Construction and expression of hybrid plasminogen activators prepared from tissue-type plasminogen activator and urokinase-type plasminogen activator genes”, J Biol Chem, vol. 263, No. 6, 1988, pp. 2917-2924.
Czugler M et al, “Noncovalent structural Imodels for the Asp-His dyad in the active site of serine proteases and for solid-state switching of protonation states”, J Am Chem Soc, 1985, vol. 108, pp. 1275-1281.
Li, Yishan et al, “Phospholipase A2 engineering. The Asp-His catalytic diad also plays an important structural role”, J Am Chem Soc, Sep. 22, 1993, vol. 115, No. 19, pp. 8523-8526.
Frey, PA, et al, “A low-barrier hydrogen bond in the catalytic triad of serine proteases”, Science, vol.264, Jun. 24, 1994, pp. 1927-1930.
Umeyama H et al, “Effects of the hydrogen bond between His57 and Asp102 on the lone pair molecular orbital of nitrogen of His57
Ahlberg Per
Baltzer Lars
A + Science Invest AB
Brusca John S.
Burns Doane Swecker & Mathis L.L.P.
Siu Stephen
LandOfFree
Acyl transfer with stabilized transition complex using... does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Acyl transfer with stabilized transition complex using..., we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Acyl transfer with stabilized transition complex using... will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-2547854